The purpose of these studies is to analyze the molecular mechanisms by which ligand occupancy of intergrin receptors and high affinity binding proteins alter the capacity of insulin-like growth factor-I (IGF-I) to stimulate smooth muscle cell (SMC) replication and migration. SMC constitutively synthesize IGF-I and IGFBPs which have been shown to modulate IGF actions. They also contain the alphaVbeta3 integrin receptor and ligand occupancy of alphaVbeta3 alters the SMC response to IGF-I. These studies to determine the role of proteolysis of IGFBP-5 in altering the amount if IGF-I is exposed to receptors. A cDNA probe containing the protease sequence and a high affinity antiprotease antiserum will be used to determine the variable that increase its synthesis and/or activation from and inactive form. They will also be used to screen for the presence of protease or inhibitors and to determine if any of the SMC growth factors that function together with IGF-1 alter protease inhibitors synthesis. The functional consequences of altering protease activity on IGF-1 actions will be determined. The role of thrombin which cleaves IGFBP-5 at physiologic concentrations or in releasing it from extracellular matrix and in modifying IGF actions will be analyzed. Additional studies will focus on the role of IGFBP-5 binding to three specific ECM proteins, thrombospondin, vitronectin and osteopontin and how this alters their ability to interact with the alphaVbeta3 receptor. Fragments of IGFBP-5 that do not bind to IGF-I will be tested for their capacity to bind to these proteins and to alter alphaVbeta3 modulation of IGF-I actions. Mutants that have been prepared that have deficient ECM binding will be analyzed to determine if binding to any of these three proteins is reduced and if selective loss of binding altered pSMC responsiveness to IGF-I. Studies will determine the mechanism by which of ligand occupancy of alphaVbeta3 modifies IGF-I receptor kinase activity will be undertaken. Studies will initiated to determine if these two receptors co-localize in the focal adhesion complex (FAC) with other FAC proteins. Since alpha2beta1 ligand occupancy is a negative regulator of IGF action, we will determine if Type IV collagen binding to this receptor modifies activation of the IGF-1 receptor by alphaVbeta3. Blocking the UPAR receptor specifically inhibits IGF-1 stimulated migration. Studies will be undertaken to determine if PI-3 kinase is an important signaling element in this pathway. The results of these studies should help to define the molecular mechanisms by which IGF-I functions coordinately with ECM proteins and integrins to stimulate SMC migration and replication and may suggest novel strategies for interfering with these processes to alter the progression of atherosclerosis.